Purification of New Peptidal Hypertensive Substances and Their Role in the Control of Blood Pressure

最近，内源性洋地黄样物质与抗地高辛抗体交叉反应，被认为是原发性高血压的可能原因，因为有报道称该物质在该病患者的血液和尿液中增加。该物质促进尿钠，抑制Na^+， K^+- atp酶，被认为可以升高血压。因此，以抑制大鼠脑Na^+， K^+ atp酶活性为指标，从高血压大鼠单侧肾脏切除后的尿液中纯化该物质，注射脱氧皮质酮醋酸酯，并给予1% NaCl溶液作为饮用水。在HCL存在下收集尿液，煮沸尿液后，采用CM-sepharose柱层析、Sephadex G-10柱层析和反相高效液相色谱进行纯化。纯化了3种对Na^+、K^+ atp酶具有抑制活性的物质。抑菌活性呈剂量依赖性，且抑菌活性不受胰蛋白酶消化的影响。但酸水解后这3种物质的活性均消失，其中2种物质经脯氨酸酶消化后活性消失。这些物质与抗地高辛抗体呈剂量依赖性交叉反应，且交叉反应与Na^+， K^+- atp酶的抑制活性平行。其中一种物质注入侧脑室后也表现出高血压作用。这些发现表明这些物质是多肽或蛋白质。对这些物质进行氨基酸分析得到氨基酸，但用氨基酸测序仪分析没有显示出任何序列信息。由于如果肽的n端被阻断，则无法确定氨基酸序列，因此我们试图去除阻断。然而，我们无法得到序列。因此，我们认为这些物质在尿液中的含量很低，我们无法获得足够的物质来分析氨基酸序列。因此，现在我们试图获得足够的物质来分析序列。在成功地确定了这些序列之后，我们将研究这些物质的生理作用。少

Recently, the endogenous digitalis like substance, which shows cross-reactivity with anti-digoxin antibody, is thought a possible cause of the essential hypertension, since this substance is reported to increase in the blood and urine of the patients of this disease. This substance enhances natriuresis, inhibits Na^+, K^+-ATPase and is considered to elevate blood pressure. Thus, using the inhibitory activity of rat brain Na^+, K^+ATPase as a marker, this substance was purified from urine of hypertensive rats which were removed of unilateral kidney, were injected wit deoxy-corti-costerone acetate and were given 1 % NaCl solution as drinking water. After collecting urine in the presence of HCL and boiling the ruine, the purification was done using CM-sepharose column chromatography, Sephadex G-10 column chromatography and reversed phase HPLC. Three substances which had inhibitory activity of the Na^+, K^+ATPase were purified. The inhibitory activities were dose-dependent, and the activit … More ies were not affected by the trypsin-digestion. however, these activities of the three substance were disappeared after the acid hydrolysis, and these of 2 substances out of the 3 were disappeared after the prolidase digestion. These substances also showed cross-reactivity to anti-digoxin antibody in dose-dependent manners and the cross-reactivity was parallel with the inhibitory activity of the Na^+, K^+-ATPase. One of the 3 substances also showed hypertensive action when it was injected into the lateral cerebral ventricle. These findings suggest that these substances are peptides or proteins. Amino acid analysis of these substances gave amino acids, but analysis using amino acid sequencer did not show any information about the sequences. Since it is impossible to determine amino acid sequence if the N-terminal of the peptide is blocked, we tried to remove the blockade. However, we could not to get the sequences. Therefore, we think contents of these substances in the urine were very low and we could not get enough substances to analyze the amino acid sequence. Thus, now we are trying to get enough substances to analyze the sequence. After succeeding to identify the sequences, we are going to examine the physiological roles of these substances. Less